1. Field of the Invention
This invention is directed to the discovery that certain peptide fragments of the prehormone, proglucagon, possess hormonal activities and can be used to stimulate the synthesis and secretion of the hormone, insulin. These peptide fragments are useful in therapy for the disease Diabetes mellitus.
2. Description of the Background Art
The endocrine secretions of the pancreatic islets are under complex control not only by blood-borne metabolites (glucose, amino acids, catecholamines, etc.), but also by local paracrine influences. The major pancreatic islet hormones (glucagon, insulin, and somatostatin) interact among their specific cell types (A, B, and D cells, respectively) to modulate secretory responses mediated by the metabolites. Although insulin secretion is predominantly controlled by blood levels of glucose, glucagon and somatostatin stimulate and inhibit glucose-mediated insulin secretory responses, respectively. In addition to the proposed interislet paracrine regulation of insulin secretion, there is evidence to support the existence of insulinotropic factors in the intestine. This concept originates from the observations that glucose taken orally is a much more potent stimulant of insulin secretion than is a comparable amount of glucose given intravenously.
The human hormone, glucagon, is a 29-amino acid peptide hormone produced in the A-cells of the pancreas. The hormone belongs to a multi-gene family of structurally related peptides that include secretin, gastric inhibitory peptide, vasoactive intestinal peptide, and glicentin. These peptides variously regulate carbohydrate metabolism, gastrointestinal mobility, and secretory processing. The principal recognized actions of pancreatic glucagon, however, are to promote glycogenolysis and gluconeogenesis, resulting in an elevation of blood sugar levels. In this regard, the actions of glucagon are counterregulatory to those of insulin and may contribute to the hyperglycemia that accompanies Diabetes mellitus (Lund, P. K., et al., Proc. Natl. Acad. Sci., USA 79:345-349 (1982)).
Glucagon has been found to be capable of binding to specific receptors which lie on the surface of insulin-producing cells. Glucagon, when bound to these receptors, stimulates the rapid synthesis of cAMP, by these cells. cAMP, in turn, has been found to stimulate insulin expression (Korman, L. Y., et al., Diabetes 34:717-722 (1985)). Insulin acts to inhibit glucagon synthesis (Review of Medical Physiology, Ganong, W. F., 1979, Lang Publications, Los Altos, California (p. 273)). Thus, the expression of glucagon is carefully regulated by insulin, and ultimately by the serum glucose level.
The glucagon gene is initially translated from a 630-base pair precursor to form the polypeptide, preproglycagon (Lund et al. (1982)). This polypeptide is subsequently processed to form proglucagon. Patzelt, C., et al. (Nature 282:260-266 (1979) demonstrated that proglucagon was subsequently cleaved into glucagon and a second polypeptide. Subsequent work by Lund, P. K., et al. (Proc. Natl. Acad. Sci. USA 79:345-349 (1982)); Lopez, L. C., et al. (Proc. Natl. Acad. Sci. USA 80:5485-5489 (1983)) and Bell, G. I., et al. (Nature 302:716-718 (1983)) demonstrated that the proglucagon molecule was cleaved immediately after lysine-arginine dipeptide residues. Studies of proglucagon produced by channel catfish (Ictalurus punctata) indicated that glucagon from this animal was also proteolytically cleaved after adjacent lysine-arginine and arginine-arginine dipeptide residues (Andrews, P. C., et al., J. Biol. Chem. 260:3910-3914 (1985)). Lopez, L. C., et al. (Proc. Natl. Acad. Sci. USA 80:5485-5489 (1983)), and Bell, G. I., et al., discovered the mammalian proglucagon was cleaved adjacent lysine-arginine or arginine-arginine dipeptides and demonstrated that the proglucagon molecule contained three discrete and highly homologous peptide molecules which were designated glucagon, glucagon-like protein 1 (GLP-1), and glucagon-like protein 2 (GLP-2). Lopez et al. concluded that glucagon-like protein 1 was 37 amino acid residues long and that glucagon-like peptide 2 was 34 amino acid residues long. Analogous studies on the structure of rat preproglucagon revealed a similar pattern of proteolytic cleavage between adjacent lysine-arginine or arginine-arginine dipeptide residues, resulting in the formation of glucagon, GLP-1, and GLP-2 (Heinrich, G., et al., Endocrinol. 115:2176-2181 (1984)). Human rat, bovine, and hamster sequences of GLP-1 have been found to be identical (Ghiglione, M., et al., Diabetoloqia 27:599-600 (1984)).
The conclusion reached by Lopez et al. (Proc. Natl. Acad. Sci. USA 80:5485-5489 (1983)) regarding the size of GLP-1 was confirmed by the work of Uttenthal, L. O., et al., (J. Clin. Endocrinol. Metabol. 61:472-479 (1985)). Uttenthal et al. examined the molecular forms of GLP-1 which were present in the human pancreas. Their research shows that GLP-1 and GLP-2 are present in the pancreas as proteins having 37 and 34 amino acid residues, respectively.
The similarity between GLP-1 and glucagon suggested to early investigators that GLP-1 might have biological activity. Although some investigators found that GLP-1 could induce rat brain cells to synthesize cAMP (Hoosein, N. M., et al., FEBS Lett. 178:83-86 (1984)), other investigators failed to identify any physiological role for GLP-1 (Lopez, L. C., et al., Proc. Natl. Acad. Sci. USA 80:5485-5489 (1983)). The failure to identify any physiological role for GLP-1 caused some investigators to question whether GLP-1 was in fact a hormone and whether the relatedness between glucagon and GLP-1 might be artifactual (Ghiglione, M., et al., Diabetologia 27:599-600 (1984)).
Thus, in conclusion, the prior art reveals an awareness of the processing of a glucagon hormone precursor into a set of peptides sharing extensive homology. It has been widely assumed by those of skill in the art that these highly related glucagon-like peptides would have a biological activity. Nevertheless, extensive investigations designed to elucidate the biological effects of these molecules had been unsuccessful.